Variations in SXT elements in epidemic Vibrio cholerae O1 El Tor strains in China

Cholera is an infectious disease caused by the gram-negative pathogen Vibrio cholerae. Cholera infection leads to severe dehydrating diarrhea. V. cholerae has caused seven disastrous pandemics in recorded history and remains a major threat to public health in developing countries. Based on the somatic O antigen, V. cholerae strains can be classified into more than 200 serogroups, although only serogroups O1 and O139 are associated with epidemic infections. The El Tor biotype of serogroup O1 is responsible for the seventh ongoing cholera pandemic and has spread throughout China since 1961. This situation continues to worsen owing to the emergence and spread of drug-resistant V. cholerae strains. Unlike the serogroup O139 strains, O1 El Tor isolates from China exhibit lower antibiotic resistance, except against nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. Interestingly, resistance to all three antibiotics has been shown to be strongly correlated with the presence of the SXT element1. The SXT element (conferring resistance to sulfa and trimethoprim) is an integrative conjugative element (ICE) belonging to the SXT/R391 family. SXT (99.5 kb in length), which was identified from a V. cholerae O139 clinical strain, MO10, isolated in India in 1992, and R391 (89.5 kb), which was identified from Providencia rettgeri, were the first reported SXT/R391 family ICEs2,3. SXT has been found in most clinical and environmental isolates of V. cholerae, including serogroup O1 from both Asian and African isolates. SXT has also been detected in other diverse species of gammaproteobacteria, such as Photobacterium damselae, Shewanella putrefaciens, and Providencia alcalifaciens4. To date, more than 30 SXT/R391 family ICEs have been sequenced. The SXT/R391 family is characterized by a conserved site-specific integrase that mediates integration into the 5′-end of the prfC gene of the host chromosome; this gene encodes peptide chain release factor 3, which is responsible for the integration and excision roles found in lambdoid phages5. SXT/R391 ICEs can be transferred between bacteria by conjugation, resulting in the transfer of a diverse array of functions to the host. Resistance to multiple antibiotics and heavy metals were the first described fitness functions in ICEs of this family6. Subsequently, functions involved in regulation of motility and biofilm formation were identified7. Elements of the SXT/R391 family share a genetic backbone of 52 syntenic genes, which mediate their integration/excision, conjugative transfer, and regulation during the life cycle of the ICEs2. In addition to this conserved core scaffold, these elements also harbor variable fragments, such as antibiotic resistance genes. The variable regions range from 30 to 60 kb in length and are mostly found at five insertion hotspots (HSs), termed HS1, HS2, HS3, HS4, and HS5, and four variable regions, named VRI, VRII, VRIII, and VRIV8. Determination of the sequences of the ICE of the SXT/R391 family is essential for elucidating their organization and evolution. Though, an elaborate ICE capture system on plasmids has been developed to facilitate their sequencing and has been used for determining five ICE sequences8, it is an arduous task due to their size and predominantly chromosomal localization. In this study, we explored the types and evolution of SXT elements in Chinese V. cholerae O1 strains by clustering the backbone genes of the SXT element. We also extracted 11 complete SXT sequences from a genomic resequencing database of V. cholerae O1 El Tor strains established in our laboratory9. Our SXT genomic structure analysis described the differences between two sequence types of SXT elements, positioned the antibiotic resistance genes on the SXT elements, and presented the key role of this ICE in the emergence and transmission of multidrug resistance in V. cholerae.